This application is directed to methods and external guide sequence compositions designed to target cleavage of RNA by RNAse P.
Ribonucleic acid (RNA) molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, but also as enzymes which specifically cleave nucleic acid molecules. Such catalytic RNA molecules are called ribozymes.
The discovery of catalytic RNA, by Drs. Altman and Cech, who were awarded the Nobel prize in 1989, has generated much interest in commercial applications, particularly in therapeutics (Altman, Proc. Natl. Acad. Sci. USA 90:10898-10900 (1993); Symons, Annu. Rev. Biochem. 61:641-671 (1992); Rossi et al., Antisense Res. Dev., 1:285-288 (1991); Cech, Annu. Rev. Biochem. 59:543-568, (1990)). Several classes of catalytic RNAs (ribozymes) have been described, including intron-derived ribozymes (WO 88/04300; see also, Cech, T., Annu. Rev. Biochem., 59:543-568, (1990)), hammerhead ribozymes (WO 89/05852 and EP 321021 by GeneShears), axehead ribozymes (WO 91/04319 and WO 91/04324 by Innovir).
RNAse P
Another class of ribozymes include the RNA portion of an enzyme, RNAse P, which is involved in the processing of transfer RNA (tRNA), a common cellular component of the protein synthesis machinery. Bacterial RNAse P includes two components, a protein (C5) and an RNA (M1). Sidney Altman and his coworkers demonstrated that the M1 RNA is capable of functioning just like the complete enzyme, showing that in Escherichia coli the RNA is essentially the catalytic component, (Guerrier-Takada et al., Cell 35:849-857 (1983)). In subsequent work, Dr. Altman and colleagues developed a method for converting virtually any RNA sequence into a substrate for bacterial RNAse P by using an external guide sequence (EGS), having at its 5' terminus at least seven nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 5' terminus the nucleotides NCCA (N is any nucleotide)(WO 92/03566 by Yale, and Forster and Altman, Science 238:407-409 (1990)).
Using similar principles, EGS/RNAse P-directed cleavage of RNA has been developed for use in eukaryotic systems, where the external guide sequence forms structures similar to the stem and loop structures of tRNA and where the substrate RNA hybridizes to ends of the EGS to form structures similar to the acceptor and D stems of tRNA (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale). It has subsequently been shown that EGS molecules for use with eukaryotic RNAse P need form only a structure similar to the T stem and loop of tRNA, where, again, the substrate RNA hybridizes to ends of the EGS to form structures similar to the acceptor and D stems of tRNA (WO 95/24489 to Yale). These EGS molecules are useful for promoting RNAse P-mediated cleavage of target RNA molecules. They are especially useful for in vivo use since only the relatively small EGS molecule need be administered. The catalytic RNAse P is already present and active in the cells of the animal or patient. Yuan and Altman, EMBO J 14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995), later determined a minimal structure necessary for cleavage of a substrate by RNase P.
It is an object of the present invention to provide a therapeutic targeted for treatment of viral diseases and diseases involving abnormal transcription products, and method of use thereof.
It is another object of the present invention to provide short external guide sequences for RNase P, vectors encoding such short external guide sequences, and methods of use thereof.
It is another object of the present invention to provide chemically modified short external guide sequences for RNAse P with enhanced resistance to nuclease degradation.
It is another object of the present invention to provide methods of cleaving target RNA molecules promoted by short external guide sequences for RNAse P.
It is a further object of the present invention to provide short external guide sequences for RNAse P specifically targeted against viruses such as hepatitis, vectors encoding such short external guide sequences, and methods of use thereof.